Articles and publications set forth in this patent disclosure are presented for the information contained therein; none of this information is admitted to be statutory "prior art" and we reserve the right to establish prior inventorship with respect to any such information.
Deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") are long, threadlike macromolecules, DNA comprising a chain of deoxyribonucleotides, and RNA comprising a chain of ribonucleotides. A nucleotide consists of a nucleoside and one or more phosphate groups; a nucleoside consists of a nitrogenous base linked to a pentose sugar. Typically, the phosphate group is attached to the fifth-carbon ("C-5") hydroxyl group ("OH") of the pentose sugar; however, it can also be attached to the third-carbon hydroxyl group ("C-3 OH"). In a molecule of DNA, the pentose sugar is deoxyribose, while in a molecule of RNA, the pentose sugar is ribose. The nitrogenous bases in DNA are adenine ("A"), cytosine ("C"), guanine ("G"), and thymine ("T"). These bases are the same for RNA, except that uracil ("U") replaces thymine. Accordingly, the major nucleosides of DNA, collectively referred to as "deoxynucleosides" are as follows: deoxyadenosine ("dA"); deoxycytidine ("dC"); deoxyguanosine ("dG"); and thymidine ("T"). The corresponding ribonucleosides are designated as "A"; "C"; "G"; and "U". (By convention, and because there is no corresponding thymidine ribonucleoside, deoxythymidine is typically designated as "T"; for consistency purposes, however, thymidine will be designated as "dT" throughout this disclosure).
The sequence of the nitrogenous bases of the DNA or RNA molecule encodes the genetic information contained in the molecule. The sugar and phosphate groups of a DNA or RNA molecule perform a structural role, forming the backbone of the molecule. Specifically, the sugar moiety of each nucleotide is linked to the sugar moiety of the adjacent nucleotide such that the 3'-hydroxyl of the pentose sugar of one nucleotide is linked to the 5'-hydroxyl of the pentose sugar of the adjacent nucleotide. The linkage between the two pentose sugars is typically via a phosphodiester bond. Based upon this linkage protocol, one end ("terminus") of the nucleotide chain has a 5'-terminus (e.g. hydroxyl, phosphate, phosphates, etc.), and the other end has e.g., a 3'-hydroxyl or phosphate group. By convention, the base sequence of a nucleotide chain is written in a 5' to 3' direction, i.e., 5'-ATCG-3', or, simply ATCG.
DNA and RNA are produced internally by living animals; however, DNA and RNA can be chemically synthesized such that synthetic strands of DNA and RNA can be rapidly and efficiently produced. These strands are typically referred to as "synthetic oligonucleotides" or "oligonucleotides." A widely utilized chemical procedure for the synthesis of oligonucleotides is referred to as the "phosphoramidite methodology." See, e.g., U.S. Pat. No. 4,415,732; McBride, L. and Caruthers, M. Tetrahedron Letters, 24.:245-248 (1983); and Sinha, N. et al. Nuc. Acids Res. 12:4539-4557 (1984), which are all incorporated herein by reference. Commercially available oligonucleotide synthesizers based upon the phosphoramidite methodology include, e.g., the Beckman Instruments OLIGO 1000; the Millipore 8750.TM.; and the ABI 380B.TM., 392.TM. and 394.TM. DNA synthesizers. Regardless of the protocol or the instrument, most typically synthetic oligonucleotides are "grown" on a support material, typically referred to as a "solid support". Solid supports are varied and well-known; specifics regarding solid supports will be set forth in detail below.
The importance of chemically synthesized oligonucleotides is principally due to the wide variety of applications to which oligonucleotides can be directed. For example, oligonucleotides can be utilized in biological studies involving genetic engineering, recombinant DNA techniques, antisense DNA, detection of genomic DNA, probing DNA and RNA from various systems, detection of protein-DNA complexes, detection of site directed mutagenesis, primers for DNA and RNA synthesis, primers for amplification techniques such as the polymerase chain reaction, ligase chain reaction, etc, templates, linkers, and molecular interaction studies. Recent attention in the area of oligonucleotide synthesis has focused on procedures generally referred to as Sequencing by Hybridization ("SBH"), as first disclosed by Edwin Southern (see European Patent Application No. WO 89/10977, "Analyzing Polynucleotide Sequences").
The primary repeating structures of DNA and RNA molecules can be depicted as the following nucleosides: ##STR1## The key step in nucleic acid synthesis is the specific and sequential formation of internucleotide phosphate linkages between a 5'-OH group of one nucleotide and a 3'-OH group of another nucleotide. Accordingly, in the typical synthesis of oligonucleotides, the phosphite group of an "incoming" nucleotide is combined with the 5'-OH group of another nucleotide (i.e. the 5'-OH group is "phosphorylated" or "phosphitylated"). These groups must be capable of actively participating in the synthesis of the oligonucleotides. Thus, the 5'-OH groups are modified (typically with a dimethoxy trityl ("DMT") group) such that an investigator can introduce two such nucleotides into a reaction chamber and adjust the conditions therein so that the two nucleotides are properly combined; by a series of successive such additions, a growing oligonucleotide having a defined sequence can be accurately generated.
Proteins and peptides are essential components of all living cells. They are the structural elements of cell walls and cell membranes, enzymes, immunoglobulins, antibodies, transport molecules and most hormones. The building blocks of proteins and peptides are the twenty natural amino acids. Each amino acid is "encoded" by the sequential grouping of three nucleotides, referred to as a "codon". Because there are four different nucleotides and three nucleotides are required to encode an amino acid, there are 64 possible codons (4.sup.3). Thus, several codons can encode for the same amino acid; for example, the codons GCG, GCA, GCT and GCC all encode for the amino acid alanine.
A series of amino acids correctly linked together via amide bonds form protein chains, and the amino acid sequence of such a protein chain ("primary structure") determines the very complex secondary and territory structures responsible for the biological functions of the proteins.
Each amino acid has an amino and carboxyl terminal, such that proteins and peptides have an amino ("N-") and a carboxyl ("C-") terminal end. The general formula of an amino acid can be depicted as follows: ##STR2## where R is one of at least 20 different side chains (for example, the side chain for alanine is a CH.sub.3 group). The "NH.sub.2 " group is the amino group, and the "COOH" group is the carboxyl group.
As with nucleic acids, synthetic linear or branched amino acid chains can be chemically synthesized. A particularly well known procedure for the synthesis of linear amino acid chains, referred to as "solid phase peptide synthesis", was introduced by Merrifield in 1963. See generally, Barany, G. and Merrifield, R. B. (1980) in The Peptides, 2:1-284. Gross, E. and Meienhofer, J. Eds. Academic Press, New York. Automated peptide synthesizers which utilize solid phase peptide synthesis protocols include, for example, the ABI 430.TM. and 431.TM. the Millipore 9050 Plus PepSynthesizer.TM., and the Milligen 9500.TM. and 9600.TM.. Typically, the C-terminal end of the first amino acid is coupled to a solid support comprising a reactive group (i.e., the site of attachment), while the N-terminal end of the first amino acid is protected with a labile protecting group (i.e., a group that can be readily removed). The side chain functional groups on the amino acids must be protected with "temporary" protecting groups. Under appropriate conditions, a similarly protected amino acid is added to the insolubilized first amino acid which has had the labile protecting group removed therefrom. By a series of successive additions, an amino acid chain can be synthesized, the final step typically being the cleavage of the chain from the solid support and the removal of the temporary protecting groups from each amino acid side chain. This leads to a biologically active protein or peptide.
Oligosaccharides are the building blocks for glycopeptides and glycolipids; glycopeptides and glycolipids can be important mediators of biological activity by interacting with cell membrane surfaces. Thus, synthetic oligosaccharides can be utilized, inter alia, to target specific cell membrane surfaces or to interfere with the natural binding of glycopeptides and glycolipids to a cell membrane surface. Synthetic oligosaccharides have recently gained notoriety for their ability to target a specific drug to a specific tissue. The solid phase synthesis of oligosaccharides has been reported using poly (ethylene glycol) monomethyl ether as the solid support. See, Douglas, S. P. et al. J. Am. Chem. Soc. 113: 5095-5097 (1991). See also, Rudemacher, T. W. et al. "Glycobiology" Ann. Rev. Biochem. 57: 785-838 (1988).
The solid supports utilized for, inter alia, nucleic acid, protein/peptide, and oligosaccharide synthesis are varied. With respect to nucleic acid synthesis, a widely utilized solid support material is controlled pore glass ("CPG"). See, for example, U.S. Pat. No. 4,458,066. Other materials include nylon, polystyrene, polyacrylamide and cellulose. Teflon.TM. fiber support has been described as a substrate for oligonucleotide synthesis. See, Lohrmann, R. A. and Ruth, J. (1984) DNA 3:122; PCT Publication WO 85/01051 (published: Mar. 14, 1985); and Molecular Biosystems, Inc. Oxidizable Solid Supports (Cat. No. OSS-01 and OSS-02). With respect to protein/peptide synthesis, such materials include, for example, cross-linked polystyrene, cellulose and polyamide resins. U.S. Pat. No. 4,923,901 describes modified membranes having bound thereto oligonucleotides and peptides. As noted, poly (ethylene glycol) monomethyl ether has been used as a solid support for oligosaccharide synthesis.
An ongoing need exists for solid supports useful in the synthesis of these types of materials. This is because the materials heretofore utilized have associated drawbacks. For example, certain supports require the use of "spacer arms" or linkers to, in effect, couple the amino acids or proteins/peptides to the solid support; typically, when such linkers are utilized, it is often necessary to block sites on the membrane where the linkers are not located, in an effect to decrease or prevent non-specific binding of the biomonomers and biopolymers to the "non-linker" locations on the support. See, for example, Zhung, Y., et al. "Single-base mutation analysis of cancer and genetic diseases using membrane bound modified oligonucleotides" Nuc. Acids Res. 19(14):3927-3933 (1991)(nylon). Other materials require the use of surface modification to graft onto the surface of the solid support an appropriate material which can in turn bind the biomonomers and biopolymers. See, for example, U.S. Pat. No. 4,923,901 (polypropylene). Still other materials require, for example, chemical modification of the support to provide the necessary linkages between the support and the biomonomers and biopolymers. See, for example, U.S. Pat. No. 4,458,066 (inorganic polymers). As is evident, these additional steps add the potential for errors, and hence can negatively impact upon positive analytical results, as well as significantly increasing the cost of the support.
What are needed, and hence, what would contribute to the state of the art, are materials which can be used for the synthesis of oligonucleotides and proteins/peptides which do not require such additional protocols such that the material is capable of being rapidly, efficiently and economically prepared. With an appropriate solid support method, oligonucleotides could be synthesized thereon, and the resulting product utilized for the analysis of patient sample DNA for determination of presence or absence of specific genetic mutations.